Summary: The long-term goal of this proposal is to characterize the mechanisms that regulate proliferation of germline stem cells (GSCs) in C. elegans. A set of genes that appear to function with known regulators of germline proliferation has been found to be necessary for setting the boundary of the GSC niche and regulating cell proliferation, including icd-1 and ced-4, known previously for their roles in apoptosis, and two gene clusters encoding micro RNAs (miRs). Several of the germline regulatory genes contain putative target binding sites for these miRs and restriction of icd-1 expression at the GSC boundary requires one cluster, suggesting direct regulatory relationships. Experiments will be performed to illuminate the action of these and other genes identified in functional genomics screens in regulating the GSC niche and cell proliferation. In the first aim, the hypotheses that germline regulating factors and several miR clusters collaborate to set the GSC boundary and regulate cell proliferation will be tested. The hypothesized role of ICD-1 as a component of a parallel pathway controlling germline proliferation, and the action of tumorous germline regulatory genes on stability of its RNA and protein abundance will be evaluated. Interactions between mutations that delete the miR clusters will be analyzed and sufficiency of the miRs and ICD-1 in controlling proliferation investigated. In the second aim, a model postulating the action of miRs and their predicted targets sites in germline regulatory genes will be tested. The distribution of miRs in the germline and their functional requirements in regulating expression of mRNAs and encoded products of these regulatory genes will be assessed. The function of predicted miR binding sites in these genes and the role of the GSC niche-determining Notch pathway in miR regulation will be examined. The third aim will apply functional genomics RNAi screens to identify new germline proliferation regulators. Computational and phenotypic studies will be used to analyze the functions of these genes in germline proliferation and their relationships to known germline regulators. These studies will help to advance our understanding of oncomirs and the processes underlying dysregulated proliferation in tumorigenesis, and mechanisms that regulate stem cell division. PUBLIC HEALTH RELEVANCE: Narrative This project analyzes how genes control the division of cells, the process that leads to formation of all human tumors. In addition, it will analyze how genes tell cells to become the right type at the proper time and place during development, which is key to avoiding human birth defects.